Glutamate is the primary excitatory neurotransmitter in the brain. Proper control of glutamatergic neurotransmission is indispensable for neuronal functions. An abnormal increase in excitability is linked to neuronal diseases such as epilepsy, Alzheimer's, and Parkinson's disease. One of the major pathways for replenishing glutamate to the presynaptic cell is the glutamine- glutamate shuttle, which consists of three steps: uptake of glutamate by glia cells, conversion of glutamate into glutamine in the glia cells, and export of glutamine back to neuronal cells. Some evidence suggesting that the glutamine-glutamate shuttle is up-regulated in pathological conditions and that the resulting increase in glutamate release might cause seizures has been reported. However, previous detection methods have been unable to achieve direct proof for the up-regulation of the glutamine-glutamate shuttle and the increase of glutamate release. We previously developed glutamate sensors that report glutamate concentration change as the change in Fluorescent Resonance Energy Transfer (FRET) efficiency between cyan and yellow fluorescent proteins. Such sensors can be expressed in living cells and report the glutamate release optically. Moreover, glutamate release from a brain slice can be detected by directly applying these sensors to the tissue, offering a method to detect glutamate transient in an intact tissue. As the next step in the detection process, we propose to develop a series of glutamine sensors to determine the effect glutamine levels have on the glutamate level in the brain. Combined with previously developed glutamate sensors, the proposed system will be useful in examining whether the disruption of the glutamine-glutamate shuttle is related to the onset of neuronal diseases. We have preliminary data to suggest that a glutamine binding protein from E. coli can be converted into a high-affinity, high-specificity FRET glutamine sensor. Using this prototype sensor as a scaffold, we will create glutamine sensors that are suitable for in vivo measurements of glutamine in the subcellular compartments that are relevant to the glutamine-glutamate shuttle. We will also further optimize the previously developed glutamate sensor for cytosolic glutamate concentration measurement and evaluate if the sensor could be used as a platform for high-throughput screening. Moreover, we propose to engineer glutamine and glutamate sensors with two spectrally orthogonal FRET paris so that we can use both sensors in the same experiment. This experimental setting will allow us to correlate glutamate and glutamine concentration change at very high time resolution. In addition, this system will serve as the proof-of-principle for dual imaging of other closely related molecules such as GABA and glutamate. PUBLIC HEALTH RELEVANCE: The control of glutamatergic neurotransmission is crucial to normal brain functions such as learning and memory, but how the level of glutamate is regulated is largely unknown. We propose to develop a novel, simultaneous detection method to detect glutamate and glutamine, the neurotransmitter and the major precursor of glutamate, in intact tissue. The result will contribute significantly to the understanding of glutamate regulation in the brain and will have a direct impact on the study of diseases that are related to glutamate excitotoxicity, such as epilepsy and Alzheimer's diseases.